Case 26

Published on 18/02/2015 by admin

Filed under Allergy and Immunology

Last modified 22/04/2025

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CASE 26

Brad, a 25-year-old man, was referred to your endocrinology service by his family physician with apparently multiple autoimmune-type manifestations. He first came to his family physician’s attention several years earlier with weight gain, listlessness, general fatigue, and coarsening of the skin. After multiple attempts to establish a diagnosis his physician finally arrived at the conclusion (seemingly confirmed by thyroid function tests) that he was hypothyroid. There was no antecedent viral illness that anyone could remember, and no other family members were affected. These problems seemed to resolve with conventional thyroid replacement therapy with levothyroxine. About 2 years later fatigue recurred despite evidence for normal thyroid replacement. Blood work at this time was suggestive of adrenocortical involvement, and indeed a diagnosis of Addison’s disease was pursued. Measurement of serum and urinary adrenocortical hormones supported this hypothesis too, and again he was treated effectively with replacement mineralcorticoid, fludrocortisone (Fluorcortef/Florinef). Once again there was no evidence of any precipitating event to this disorder (drugs/infection) and no family members were affected. Finally, just 3 weeks ago he presented with an oral Candida albicans infection, which has been inordinately resistant to topical antifungal agents, and his physician is at a total loss. He is essentially unsure whether Brad is just “unlucky” or whether all of these events are tied together and are independent manifestations of some disorder he has not heard of. The answer is in fact likely to be the latter.

QUESTIONS FOR GROUP DISCUSSION

1. Autoimmune polyendocrinopathy syndrome (APS-1) has been mapped to a unique gene, AIRE (AutoImmune REgulator), on chromosome 21. What is the role of the protein that it encodes?
2. What is the criteria for a diagnosis of APS-I? Of APS-II? What technique is used to confirm a mutation in the AIRE gene?
3. Review the role of the thymus in tolerance induction (see Case 2 and the discussion on positive and negative selection).
4. Explain the significance of the fact that an equivalent gene in the mouse has been shown to be expressed in the thymus at sites where negative selection occurs (see Case 2).
5. What is the significance of the fact that a number of organ-specific antigens are actually now known to be expressed in the thymus (e.g., insulin, myelin/oligodendrocyte glycoprotein (MOG), proteolipid protein (PLP), and glutamic acid decarboxylase)?
6. Knockout mice for the AIRE gene equivalent do not express the AIRE equivalent protein. Explain the significance of the fact that they have multiple organ-specific immune syndromes (e.g., orchitis, retinitis), and have antibody titers to multiple organ-specific antibodies.
7. The knockout mice (question 4) have no obvious immune cell abnormalities when compared with wild-type mice. However, there is a subtle increase in thymic epithelial cells in the medulla, as determined using CD80 (B7-1) and CR-1 (antibody specific to thymic epithelial cells). What is the role of CD80 in T cell activation?
8. The knockout mice (question 4) also had an increased number of activated cells in the periphery as determined using CD44 (high) and CD62L (low). CD44 is a glycoprotein that binds to hyaluronan. CD62L is also known as l-selectin, an adhesive molecule that binds to carbohydrates on the endothelium to induce cell rolling. What is the significance of an increase in activated T cells in the periphery in these knockout mice?
9. In further studies with the knockout mice (question 4), thymuses were removed and treated to remove mature T cells. Then these thymuses were transferred into athymic mice (nu/nu); the mice all have the AIRE phenotype described in question 4. On the basis of these studies, hypothesize a role for the AIRE gene in tolerance induction.
10. Why could negative selection of T cells specific for organ-specific genes not occur in the absence of promiscuous transcription of those genes in the medulla?

RECOMMENDED APPROACH

Implications/Analysis of Family History

No other affected family members were identified. This does not rule out a genetic disorder in that diseases that have an autosomal recessive inheritance are quite rare. In this type of inheritance disease manifestation occurs only when the individual inherits two defective copies of the gene.

Implications/Analysis of Clinical History

There was no antecedent viral illness that anyone could remember, nor was there any evidence of any precipitating event to this disorder (drugs/infection). The weight gain, listlessness, general fatigue, and coarsening of the skin are symptoms that present in a patient whose thyroid is underactive. The presentation of Addison’s disease in a patient who is receiving therapy for hypothyroidism should have steered the physician into considering disorders that affect multiple endocrine systems, in particular autoimmune polyendocrine syndrome (APS).

APS is classified as either APS-I or APS-II. The APS-I classification applies to those syndromes that include Addison’s disease, autoimmune thyroid dysfunction, and chronic mucocutaneous candidiasis. The APS-II classification differs from the APS-I in that these patients present with type 1A diabetes instead of mucocutaneous candidiasis (along with Addison’s disease and autoimmune thyroid dysfunction). The fact that Brad had recently presented with a Candida albicans infection that was inordinately resistant to topical antifungal agents, in addition to the other endocrine disorders, strongly suggests that Brad has APS-I.

Implications/Analysis of Laboratory Investigation

The laboratory investigations over the past few years had confirmed a diagnosis of hypothyroidism, Addison’s disease, and now Candida albicans infection.

Additional Laboratory Tests

Patients with APS have mutations in the AIRE (AutoImmune REgulator) gene, which maps to human chromosome 21 (see Etiology). Polymerase chain reaction (PCR) and sequencing are used to confirm a mutation in the AIRE gene.

DIAGNOSIS

Brad was diagnosed with the autoimmune polyendocrinopathy syndrome, type I, which is a rare autoimmune disease with autosomal recessive inheritance. Patients with APS-I present with hypothyroidism, Addison’s disease, and candidiasis.

THERAPY

Continue with levothyroxine and fludrocortisone. Treat infections as they arise.

ETIOLOGY: AUTOIMMUNE POLYENDOCRINOPATHY SYNDROME

APS-I has been mapped to a unique gene, AIRE, which maps to human chromosome 21. More than 42 different mutations in the AIRE gene have been described. The AIRE gene encodes a transcription factor for many genes. Immunologic tissues, particularly the thymus, show high expression of AIRE. The high expression of the AIRE gene in the thymus, combined with the widespread autoimmune disorders that develop in mice in which this gene has been knocked out, has led to the proposal that the normal AIRE gene encodes a protein that plays a significant role in tolerance induction during T cell development.

In particular, it has been proposed that the AIRE gene product enhances the ectopic expression of peripheral tissue proteins within the thymus (medullary epithelial cells), the site of negative selection during central tolerance induction. It follows, therefore, that self tolerance to peripheral tissue antigens would not occur in the absence of a functional AIRE gene because promiscuous transcription of these peripheral proteins would either not occur or they would be expressed at only low levels in the thymus. The diseases represented in APS-I are presumably those (multiple ones) whose proteins escaped tolerance induction by virtue of low level (or absence of) thymic (medulla) expression. In the absence of this expression, autoreactive T cells are not deleted and tolerance induction does not occur, which leads to multiple autoimmune disorders.

A number of known organ-specific antigens are now known to be ectopically expressed in the presence of a normal AIRE protein (e.g., myelin/oligodendrocyte glycoprotein [MOG], proteolipid protein [PLP], insulin, and glutamic acid decarboxylase), particularly in the medulla, where negative selection occurs.

Murine Studies

An equivalent gene has been discovered in the mouse (˜80% homology at protein level). The mouse gene encodes a protein, some 540 amino acids, made up of 14 exons. It is expressed in the thymus, in the medullary epithelium, in dendritic cells, and in essentially all sites of negative selection.

Knockout Mice

A homologous deletion mutant for the mouse gene (knocking out exon 2, which introduces a frameshift and early termination, with no detectable protein) leads to multiple organ-specific immune syndromes (e.g., orchitis, retinitis) in association with development of multiple organ-specific antibodies, much like APS.

Comparison of Knockout Mice with Wild-Type Mice

The knockout mouse has no other obvious abnormalities. It has equivalent numbers to wild-type mice of CD4+/CD8+, αβTCR+, γδTCR+, CD69+ cells, and the same number and function of B cells; its polymorphonuclear cells are equivalent to those of wild-type mice, as are the splenic dendritic cells and antigen-presenting cells. Mixed lymphocyte responses (MLRs) are equivalent, and there is the same cytokine profile after concanavalin A stimulation. There is a subtle increase in thymic epithelial cells in the medulla, marked by CD80 (B7-1) and CR-1, and an increased number of activated T cells in the periphery (CD44hi, CD62Llo [l-selectin]).

Role of the Thymus in the AIRE Phenotype

In an attempt to understand the human disorder, thymuses were removed from the knockout mice, depleted of all mature lymphocytes with 2-deoxyguanosine in vitro (thus eliminating the possibility of transferring known immunoregulatory CD4+CD25+ Treg), and transplanted to nu/nu (athymic) mice. At 8 weeks, all mice have the AIRE phenotype. When detailed molecular biology comparisons were performed by gene chip expression in the thymic medullary epithelium of wild-type and knockout mice, some 200 to 1000 genes were found to be affected.

Survival of Autoreactive T Cells

A number of organ-specific tissue-antigen encoding genes are downregulated in knockout mice (e.g., insulin but not glutamic acid decarboxylase). Thus, one possible explanation for these data hypothesizes that AIRE protein enhances organ-specific antigen expression in thymus. Failure to express those organ-specific antigens (in the thymic medulla during development of the T cell repertoire) results in failure to delete autoreactive T cells, tolerance induction does not occur, and this results in multiple autoimmune diseases.

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